Cryofixation is a sample preparation method often used in electron microscopy. In this, a water-containing sample is frozen very rapidly to a temperature below −150° C. (cryofixed), i.e. it is cooled very quickly while avoiding the formation of ice crystals. Cryofixation has proven to be particularly suitable for investigations of structural biology. The specimens to be investigated, for example cells, enzymes, viruses, or lipid layers, are thereby embedded in a thin, vitrified layer of ice. The great advantage of cryofixation is that the biological structures can be maintained in their natural state. For example, a biological process can be halted at any arbitrary point in time by cryofixation and investigated in that vitrified state, for example using a cryo-electron microscope and/or in a light microscope with corresponding sample cooling. Correlative methods between a light microscope and electron microscope, also referred to as “CLEM” (correlative light-electron microscopy), make it possible, for example, firstly to observe a biological sample in a light microscope until the desired state is reached. The sample is then transferred into a cryopreparation apparatus and cryofixed for electron microscopy observation. In another variant of CLEM, the light-microscopy investigation is performed on the already cryofixed sample. Cryofixed samples can furthermore also be subjected, in a manner known per se, to further preparation steps, for example processing using freeze-fracture technology (freeze-etching) and/or coating techniques.
In order not to impair the quality of the frozen samples, it is very important that they be transferred in cooled and contamination-free fashion between the processing devices being used (for example cryofixation device, freeze fracture apparatus, coating apparatus), and the analysis devices (e.g. cryo-electron microscope, cooled light microscope).
The brochure for the “Leica EM VCT100” vacuum cryo-transfer system (manufacturer: Leica Microsystems), which is accessible via the link http://leica-microsystems.com/fileadmin/downloads/Leica%20EM%20VCT100/Brochures/Leica_EMVCT100_Brochure_EN.pdf, discloses a liquid nitrogen-cooled loading station to which a transfer container (Leica EM VCT100 Shuttle) can be coupled. A sample holder is detachably fastenable to a slide rod of the transfer container. The sample holder can be transferred out of the cooled transfer container into a cooled chamber of the loading station by displacement of the slide rod. A receptacle for retaining the sample holder is arranged inside the chamber of the loading station. The very small frozen electron microscopy samples, which are usually located in a manner known per se on an electron microscopy sample carrier (e.g. a grid or a pin for scanning electron microscopy), are manually introduced into the liquid nitrogen-cooled chamber of the loading station. The sample carrier having the sample is removed, for example with a forceps, and fastened in the sample holder. This process occurs in a cooled state, so that the frozen sample does not thaw or melt and thus become unusable. The transfer container having the sample holder and the sample carrier with a sample is then uncoupled from the loading station and attached to a corresponding apparatus (e.g. freeze fracture apparatus, cryo-electron microscope) for further processing or analysis.
The number and capabilities of sample processing operations, analyses, and corresponding devices in electron microscopy is constantly increasing. Translocation of the sample into a differently configured sample holder, or into a differently configured sample transfer device, is normally also necessary for the various applications. With the known loading station described above, translocation of a sample from one sample holder into a differently configured sample holder for a different application is not possible. It is also possible to attach only a single transfer container. Users therefore usually make do by transporting the sample to various installation sites in small containers and in liquid nitrogen. This not only involves a considerable expenditure of time, but encompasses critical working steps in which the samples can become damaged or contaminated. The working step in which the error occurred is then often not perceptible. The known loading station furthermore has no temperature monitoring, and coolant replenishment occurs manually.